toxins Article Characterization of Two Novel Bacillus thuringiensis Cry8 Toxins Reveal Differential Specificity of Protoxins or Activated Toxins against Chrysomeloidea Coleopteran Superfamily Changlong Shu 1, Guixin Yan 1, Shizhi Huang 1, Yongxin Geng 1, Mario Soberón 2 , Alejandra Bravo 2 , Lili Geng 1 and Jie Zhang 1,* 1 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; [email protected] (C.S.); [email protected] (G.Y.); [email protected] (S.H.); [email protected] (Y.G.); [email protected] (L.G.) 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62250, Mexico; [email protected] (M.S.); [email protected] (A.B.) * Correspondence: [email protected]; Tel.: +86-10-62812642 Received: 14 September 2020; Accepted: 2 October 2020; Published: 5 October 2020 Abstract: Scarabaeoidea and Chrysomeloidea insects are agriculture-destructive coleopteran pests. Few effective Bacillus thuringiensis (Bt) insecticidal proteins against these species have been described. Bt isolate BtSU4 was found to be active against coleopteran insects. Genome sequencing revealed two new cry8 genes in BtSU4, designated as cry8Ha1 and cry8Ia1. Both genes expressed a 135 kDa protoxin forming irregular shape crystals. Bioassays performed with Cry8Ha1 protoxin showed that it was toxic to both larvae and adult stages of Holotrichia parallela, also to Holotrichia oblita adults and to Anoplophora glabripennis larvae, but was not toxic to larval stages of H. oblita or Colaphellus bowringi. The Cry8Ia1 protoxin only showed toxicity against H. parallela larvae. After activation with chymotrypsin, the Cry8Ha1 activated toxin lost its insecticidal activity against H. oblita adults and reduced its activity on H. parallela adults, but gained toxicity against C. bowringi larvae, a Chrysomeloidea insect pest that feeds on crucifer crops. The chymotrypsin activated Cry8Ia1 toxin did not show toxicity to any one of these insects. These data show that Cry8Ha1 and Cry8Ia1 protoxin and activated toxin proteins have differential toxicity to diverse coleopteran species, and that protoxin is a more robust protein for the control of coleopteran insects. Keywords: Bacillus thuringiensis; cry8 genes; Coleopteran; insecticidal activity; Cry protoxin; dual mode of action Key Contribution: The study shows that Cry8Ha1 protein has a broad insecticidal spectrum against Chrysomeloidea and Scarabaeoidea pests. 1. Introduction The Coleoptera (beetles) insect order contains more described species than any other animal group, with over 380,000 named species [1,2], comprising almost 40% of described insects and nearly 30% of all animal species. Among these beetles, the species from Scarabaeoidea and Chrysomeloidae superfamilies are considered as the most destructive coleopteran pests, which cause severe damages in agriculture, horticulture, and forestry. The insecticide formulations based on Bacillus thuringiensis (Bt) insecticidal proteins, as well as transgenic plants expressing Bt insecticidal proteins are applied as Toxins 2020, 12, 642; doi:10.3390/toxins12100642 www.mdpi.com/journal/toxins Toxins 2020, 12, 642 2 of 11 sustainable control methods for some species of these coleopteran pests [3,4]. However, comparing to the beetle’s diversity, the currently discovered variability of Bt insecticidal proteins effective against coleopteran insects is scarce. Currently, only few members in Cry1, Cry3, Cry6 (now APP6), Cry7 Cry8, Cry22 (now XPP22), Cry23/Cry37 (now MPP23/XPP37), Cry34/Cry35 (now GPP34/TPP35), Cry55 (now XPP55), Cyt1, Cyt2, Sip (now MPP5), and Vip1/Vip2 subgroups of Bt protein families were reported to be toxic against certain coleopteran insects [5,6]. Hence, screening of Bt strains and identification of novel insecticidal proteins active against coleopteran insects, especially proteins that display a broad insecticidal spectrum against Chrysomeloidea and Scarabaeoidea is still needed for the effective control of these pests. Bt insecticidal crystal proteins (ICPs) are synthetized as protoxins which, once ingested by the insect, are required to be solubilized, and proteolytically activated in the insect midgut [7,8]. The activated toxin binds to membrane receptors and lyses midgut epithelial cells by forming pores that cause cell swelling. Some of the Cry toxins with potential activity are only toxic after in vitro solubilization and activation with commercial proteases. For example, the toxicity of Cry7Aa towards coleopteran insect larvae was revealed only after an in vitro solubilization and activation with proteases [9]. For this reason, it is recommended to perform bioassays with both protoxins and activated toxins to detect insecticidal activity of a new Bt ICPs, especially against coleopteran larvae. In this report, we screened a new Bt strain, BtSU4, with insecticidal activity against Holotrichia parallela and we cloned two novel cry8-type genes from this strain. Furthermore, the expression, solubilization, activation and toxicity assays of the two novel ICPs indicated that the new Cry8Ha1 protein has a broad insecticidal spectrum important for the control of several important coleopteran pests. 2. Results 2.1. Cloning and Sequence Analysis of cry8Ha1 and cry8Ia1 from BtSU4 After BtSU4 genome sequencing and insecticidal gene annotation, two genes coding for proteins with similarity to Cry8 family were discovered. The two Cry8 proteins consisted of 1199 and 1194 amino acids, respectively. The two novel proteins were subsequently named as Cry8Ha1 and Cry8Ia1 (https://www.bpprc.org). After CDD search, the core region of the two novel insecticidal proteins were identified. The domains I, II, and III of Cry8Ha1 protein were located from amino acids 94 to 290, 300 to 510 and 512 to 657, respectively. While for Cry8Ia1 protein these domain regions were located from amino acids 106 to 288, 296 to 516, and 517 to 665, respectively. BLAST analysis results showed that some domains of the two Cry8H proteins have high similarity to other proteins from the Cry family such as Cry3, Cry1B, or Cry1I, rather than to Cry8 members. Phylogenetic analysis were performed with the complete protoxin or the toxin core sequences of these two Cry8 proteins and compared with different Cry proteins (Figure1). These analyses showed that protoxin sequences of Cry8Ha1 and Cry8Ia1 clustered in a single branch with the other Cry8 protoxins as expected. In contrast the sequence of the toxin cores of these proteins were found in different branches. The toxin core of Cry8Ha1 toxin was found clustered in the same branch with Cry3 proteins together with Cry8Ba, Cry8Pa, Cry8Ca, and Cry8Ja, while Cry8Ia1 was found grouped with Cry8G, Cry8K, and Cry7G toxins (Figure1). Suggesting that these two novel proteins Cry8Ha1 and Cry8Ia1 may have differential specificity. We also performed phylogenetic analyses of the different domains. Analysis of domain I sequences from several Cry proteins showed that this region is distributed in three main clusters (Figure S1). Cluster A includes 11 different Cry8 proteins clustered with domain I sequences from Cry1B and Cry1I (Figure S1, Cluster A); the second group (Figure S1, Cluster B), includes domain I sequences from Cry8Ha1 and Cry8Ia1 that were found to be grouped with other nine Cry8 and Cry3 proteins; the third group (Figure S1, Cluster C) includes six members, forming a cluster including more distant Cry8 proteins (Figure S1). The sequences from domains II and III showed larger variation, where Toxins 2020, 12, 642 3 of 11 the Cry8Ha1 and Cry8Ia1 were located in different clusters. For domain II sequences six different clusters were identified, the Cry8Ha1 was located in Cluster A (Figure S2, Cluster A), which included sequences of domain II from Cry1B and Cry1I proteins, while Cry8Ia1 is found in cluster E with other Cry8 members such as Cry8Ib1, Cry8Ga1, Cry8Ka1, and Cry8Kb1 (Figure S2, Cluster E). Finally, for domain III sequences, they were classified in seven clusters, where the domain III of Cry8Ha1 is found in Cluster A, which includes sequences from Cry1I, Cry1B and Cry3 proteins (Figure S3, Cluster A), while Cry8Ia1 is found in Cluster E together with other Cry8 proteins, such as Cry8Ib1, Cry8Sa1, Cry8Ta,1 and Cry8La1 (Figure S3, Cluster E). These analyses show that Cry8Ha1 toxin is more closely relatedToxins with 2020 Cry3,, 12, x FOR Cry1B, PEER REVIEW and Cry1I. 5 of 11 FigureFigure 1. Phylogenetic 1. Phylogenetic analysis analysis of of the the protoxinprotoxin or or toxi toxinn core core amino amino acid acid sequences sequences from from different diff erentCry Cry proteins.proteins. These These analyses analyses involved involved 52 52 amino amino acid se sequences.quences. Evolutionary Evolutionary analyses analyses were were conducted conducted in in ClustalClustal Omega Omega and and Neighbour-joining Neighbour-joining Phylogenetic Phylogenetic analysis analysis (Madeira (Madeira et al. et al.2019). 2019). The TheCry8Ha1 Cry8Ha1 and and Cry8Ia1 proteins were labeled in red letters. Cry8Ia1 proteins were labeled in red letters. 2.2. DifferentialWe also Toxicity performed of Cry8Ha1 phylogenetic and Cry8Ia1 analyses Protoxins of the anddifferent Activated domains. Toxins Analysis Against of Di ffdomainerent I Coleopteransequences Insects from several Cry proteins showed that this region is distributed in three main clusters (Figure S1). Cluster A includes 11 different Cry8 proteins clustered with domain I sequences from Cry1BTo